1 general consideration about gene expression and expression studies expression studies expression...

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General consideration about Gene Expression and expression studies

• Expression studies

• Expression Host -> Expression System

• Promoter system -> expression vector

• Properties of product -> stability

• Production level

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Expression studies

1. Analyzing Transcription - Northern blot - Micro array - real-time PCR - Primer extension

2. Promoter studies Use of report genes to study regulatory elements

3. Analyzing Translation - Western blot - immuno assays - 2D electrophoresis - proteomics

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Northern Blot

-> to study transcription level

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Studying Transcription

Microarray technique – DNA chips

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Studying Transcription

Microarray technique – DNA chips

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Microarray technique

to study upregulation and downregulation of gene expression

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Studying Transcription

Primer Extension

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Promoter Studies

Used reporter genes: - Lac Z- GFP- Luciferase

Promoter

Reporter gene

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Promoter studies by using reporter genes

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Use of green fluorescent protein (GFP) as a reporter gene.

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Analyzing Translation – Western Blot

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2 D Electrophoresis

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Comparison of expression systems

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Prokaryotic Expression vector

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Eukaryotic Expression vector

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-> mainly in prokaryotes (E. coli)-> 2µ plasmid in yeast-> some virus vectors in mammalien cells

-> mainly in eukaryotes-> some bacterial systems (Bacillus)

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Gene Expression

Transcriptional start

Translational start

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Principal factors in bacterial expression

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Type of expression vectors

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Prokaryotic Translational vector

PstI

T7/lac

phoA cutinase

NarISalI HindIII

BamHI

phoA-cut

NdeI

S/D Term

pFCEX1

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Gene Expression

Gene copy number:

1. Plasmid copy number:

The copy-number of a plasmid in the cell is determined by regulating the initiation of plasmid replication.

The initiation of plasmid replication may be controlled by:

- the amount of available primer (RNA)

- the amount of essential replication proteins

- the function of essential replication proteins.

2. Gene dosage -> number of genes integrated into chromosome

- prokaryotic systems -> i.e. Transposons, phages, recombinantion

- mainly eukaryotic systems

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Incompatibility of plasmids:

Not all plasmids are able to coexist in the same cell. Plasmids which have the same replication control functions are incompatible, and are assigned to the same incompatibility group (inc group). Plasmids of one incompatibility group are related to each other, but cannot survive together in the same bacterial cell, as only different kinds of plasmids are compatible.

Ensures that we can make libraries -> just one plasmid taken up by one cell

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Homologous integration into chromosome

Insertion on Bacillus subtilis chromosome

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mRNA stability

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Translation Initiation

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Codon Usage of Organisms

Rare codons -> pauses translation -> lower production level

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Fusion proteins

• increase production level

• facilitate purification (taq)

• detection of expression (GFP fusion)

• Redirection of proteins (secretion -> signal

peptidases)

• Surface display (for screening of libraries)

• Tandem arrays (for small peptides, toxic proteins,..)

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Some problems of production in E. coli

31Electron micrograph of an inclusion body of the protein prochymosin in an E. coli cell

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Protein Folding

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Some E.coli expression host considerations

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Secretion of Protein

-> to avoid inclusion body formation at high expression level

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